RESEARCH ARTICLE Open Access
Oxida i e damage and esponse o Bacillus
Calme e-Gué in in bladde cance cells
exp essing sialyl ans e ase ST3GAL1
Paulo F. Se e ino
1,3†
, Ma iana Sil a
1†
, Mylene Ca ascal
1
, Nadia Malagolini
3
, Ma iella Chi icolo
3
, Giulia Ven u i
3
,
Robe o Ba ba o Fo leo
3
, Annalisa As ol i
4
, Ma iangela Ca e a
3
, Paula A. Videi a
1,2*
and Fabio Dall’Olio
3*
Abs ac
Backg ound: T ea men wi h Bacillus Calme e-Gué in (BCG) is he gold s anda d adju an immuno he apy o non-
muscle in asi e bladde cance (NMIBC), al hough i ails in one hi d o he pa ien s. NMIBC exp esses wo umo -
associa ed O-linked ca bohyd a es: he disaccha ide (Galβ1,3GalNAc) Thomsen-F ieden eich (T) an igen, and i s
sialyla ed coun e pa (Siaα2,3Galβ1,3GalNAc) sialyl-T (sT), syn hesized by sialyl ans e ase ST3GAL1, whose oles in
BCG esponse a e unknown.
Me hods: The human bladde cance (BC) cell line HT1376 s ongly exp essing he T an igen, was e o i ally
ansduced wi h he ST3GAL1 cDNA o wi h an emp y ec o , yielding he cell lines HT1376
sT
and HT1376
T
, ha
exp ess, espec i ely, ei he he sT o he T an igens. Cells we e in i o challenged wi h BCG. Whole gene exp ession
was s udied by mic oa ay echnology, cy okine sec e ion was measu ed by mul iplex immune-beads assay. Human
mac ophages de i ed om blood monocy es we e challenged wi h he sec e ome o BCG-challenged BC cells.
Resul s: The sec e ome om BCG-challenged HT1376
sT
cells induced a s onge mac ophage sec e ion o IL-6, IL-1β,
TNFαand IL-10 han ha o HT1376
T
cells. T ansc ip omic analysis e ealed ha ST3GAL1 o e exp ession and T/sT
eplacemen modula ed hund eds o genes. Se e al genes p ese ing genomic s abili y we e down- egula ed in
HT1376
sT
cells which, as a consequence, displayed inc eased sensi i i y o oxida i e damage. A e BCG challenge, he
ansc ip ome o HT1376
sT
cells showed highe suscep ibili y o BCG modula ion han ha o HT1376
T
cells.
Conclusions: High ST3GAL1 exp ession and T/sT eplacemen in BCG challenged-BC cance cells induce a s onge
mac ophage esponse and al e he gene exp ession owa ds genomic ins abili y, indica ing a po en ial impac on BC
biology and pa ien ’s esponse o BCG.
Keywo ds: Bacillus Calme e-Gué in, Glycosyla ion, Sialyl T an igen, Sialyl ans e ase, Thomsen-F ieden eich an igen
Backg ound
The in a esical inocula ion wi h he Bacillus Calme e-
Gué in (BCG) is he mos e ec i e adju an he apy o
non-muscle in asi e bladde cance s (NMIBC) a e
ansu e h al esec ion. Howe e , one hi d o he
pa ien s ail o espond and expe ience ecu ence a e
ea men . The easons why BCG he apy ail a e s ill
unclea , al hough i is well es ablished ha he an i-
umo ac i i y o BCG depends on i s abili y o elici an
e ec i e local immune esponse [1–3].
Glycosyla ion, one o he mos equen pos - ansla ional
modi ica ion o p o eins, unde goes p o ound changes in all
ypes o cance [4], including bladde cance (BC) [5–8]. The
abe an exp ession o glycoconjuga es is o en caused by he
de anged egula ion o hei biosyn he ic enzymes: he glyco-
syl ans e ases [9]. The Thomsen-F ieden eich (T) an igen is
a disaccha ide (Galβ1,3GalNAc) O-linked o se ine o h eo-
nine esidues o glycop o eins (Addi ional ile 1A), whose
* Co espondence: [email p o ec ed];[email p o ec ed]
†
Equal con ibu o s
1
Cen o de Es udos de Doenças C ónicas, CEDOC, NOVA Medical School/
Faculdade de Ciências Médicas, Uni e sidade NOVA de Lisboa, Campo dos
Má i es da Pá ia, 130, 1169-056 Lisbon, Po ugal
3
Dipa imen o di Medicina Specialis ica, Diagnos ica e Spe imen ale, Sede di
Pa ologia Gene ale, Uni e si à di Bologna, Via S. Giacomo 14, 40126 Bologna,
I aly
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Se e ino e al. BMC Cance (2018) 18:198
h ps://doi.o g/10.1186/s12885-018-4107-1
abe an exp ession in cance has been associa ed wi h ma-
lignancy [10–13] and used as a possible a ge o he apy
[14–16]. I s sialyla ed coun e pa , he sialyl-T (sT) (Siaα2,3-
Galβ1,3GalNAc-O-Se /Th ) s uc u e and i s main biosyn-
he ic enzyme, he sialyl ans e ase ST3GAL1, a e also
abe an ly exp essed in a a ie y o cance s [17,18]
[ e iewed in [19,20]]. In BC, he exp ession o T/sT an igens
is also abe an and i in luences in asion and immune ec-
ogni ion [21–23]. In a p e ious wo k [24], we ha e shown
ha he mRNA o ST3GAL1 was o e exp essed in NMIBC
bu no in muscle in asi e BC o in benign bladde umo s
and ST3GAL1 plays he majo ole in he sialyla ion o he T
an igen in BC. The T an igen has been sugges ed as a use ul
ma ke o BCG esponse [23], e en hough he ela ionship
be ween ST3GAL1/sT and BCG esponse has ne e been
es ablished.
In his s udy, we in es iga ed he e ec s o he al e na-
i e exp ession o he T o sT an igens on he abili y o
BC cells o ac i a e mac ophages in esponse o BCG
challenge and on he ansc ip ome o BC cells, u ilizing
he HT1376 cell line in which he T an igen was eplaced
by he sT an igen, by e o i al ansduc ion wi h he
ST3GAL1 cDNA. This cell line was chosen because o
i s low ST3GAL1 exp ession and i s high and
homogenous eac i i y wi h he T an igen-speci ic lec in
PNA [24]. The gene exp ession and cy okine p o iles o
he cell lines exp essing ei he he T o he sT an igens
a e BCG challenge and he abili y o hei sec e ome o
s imula e cy okine elease by mac ophages was s udied.
Me hods
Gene a ion o ST3GAL1-exp essing cell lines
The HT1376 cell line was es ablished om a p ima y in-
asi e ansi ional cell cance o he bladde [25]. Cells
we e g own in DMEM (4.5 g/L glucose, Sigma), con ain-
ing 10% oe al cal se um (FCS, Sigma), 2 mM L-glu am-
ine (Sigma) and 100 μg/mL penicillin/s ep omycin
(Sigma). HT1376 cells exp essing ST3GAL1 we e gene -
a ed by ansduc ion wi h a e o i al ec o ob ained
wi h he Vi aPowe Len i i al Exp ession Sys em (In i-
ogen), acco ding o manu ac u e ’s ins uc ions. The
cDNA o he whole coding egion o human ST3GAL1
was ob ained by PCR ampli ica ion o he cDNA o he
colon cance cell line HT29 wi h he ollowing p ime
pai : o wa d p ime : 5’-CACCATGGTGACCCTGCG-
GAAGAGG-3′; e e se p ime : 5’-TCATCTCCCCTT-
GAAGATCCGG-3′. Ampli ica ion was pe o med o
35 cycles o he ollowing p og am: dena u a ion 94 °C
1 min; annealing 60 °C 1 min; ex ension 72 °C 1 min.
The PCR p oduc was gel isola ed and cloned in o he
pLen i6/V5 Di ec ional TOPO cloning ec o (In i o-
gen) which d i es he exp ession o inse ed genes unde
he con ol o he cy omegalo i us p omo e . A nega i e
con ol e o i al ec o was p epa ed wi h an emp y
plasmid. A e ansduc ion wi h nega i e con ol- o
ST3GAL1-exp essing ec o s, HT1376 cells we e selec ed
wi h 4 μg/mL o blas icidin. The eplacemen o he T
an igen wi h he sT an igen was e alua ed as loss o cell
eac i i y wi h he luo escen labeled lec in om A achis
hypogea (peanu agglu inin, PNA), conjuga ed wi h luo -
escein iso hiocyana e (PNA-FITC). Al hough selec ed cells
we e mainly nega i e o PNA-FITC as de ec ed by FACS
analysis, a small popula ion o PNA-FITC posi i e cells
was s ill p esen . To ob ain a popula ion o cells homoge-
neously nega i e o he T an igen, abou 100 ST3GAL1-
ansduced HT1376 cells we e seeded in a 10 cm Pe i
dish and a e one mon h, PNA-FITC nega i e colonies
we e selec ed and pooled. This polyclonal cell popula ion
homogeneously nega i e o T an igen exp ession is he e-
a e e e ed o as HT1376
sT
. The polyclonal cell popula-
ion ob ained a e ansduc ion wi h he nega i e con ol
e o i al ec o ollowed by blas icidin selec ion is e-
e ed o as HT1376
T
.
Flow cy ome y
Cells we e incuba ed wi h PNA-FITC o 30 min a 4 °C
in he da k, washed and analyzed by low cy ome y. Si-
alidase ea men was pe o med wi h 20 mU o Clos-
idium pe ingens sialidase (Roche Diagnos ics), o
90 min a 37 °C.
Real ime RT-PCR
To al RNA was isola ed using he GenElu e Mammalian
To al RNA Pu i ica ion ki and DNase ea men
(Sigma), acco ding o he manu ac u e ’s ins uc ions.
One mic og am o o al RNA was e e se ansc ibed,
using he andom-p ime s based High Capaci y cDNA
A chi e Ki (Applied Biosys ems). The exp ession le el
o ST3GAL1 (Hs00161688_m1; NM_173344.2 and
NM_003033.3) was e alua ed wi h he TaqMan assay
sys em in a 7500 Fas Real-Time PCR Sys em (Applied
Biosys ems) using he TaqMan Uni e sal PCR Mas e
Mix Fas , as p e iously desc ibed [24,26,27]. The e i-
ciency o he ampli ica ion eac ion o each p ime -p obe
was abo e 95% (as de e mined by he manu ac u e ). No -
malized mRNA exp ession was compu ed as he numbe
o mRNA molecules o he gene o in e es pe 1000
mRNA molecules o he endogenous con ol β-ac in gene,
calcula ed using he 2
-ΔCT
×1000 o mula [28].
Sialyl ans e ase ac i i y assay
Cell pelle s we e homogenized in wa e and he p o ein
concen a ion o he homogena es was de e mined by
he Low y me hod. The ac i i y o ST3GAL1 was
measu ed in he homogena es in he ange o ime and
subs a e concen a ion linea i y in a 25 μL olume con-
aining: 50 mM o 2-(N-mo pholino)e hanesulphonic
acid (MES) bu e pH 6.5, 0.5% T i on X-100, 23.5 μgo
Se e ino e al. BMC Cance (2018) 18:198 Page 2 o 9
Galβ1,3GalNAcα1-O-benzyl (benzyl-T; Sigma) as ac-
cep o subs a e, 15 μM (640 Bq) o CMP-[
14
C]Sia
(Ame sham) and 50 μg o homogena e p o eins. Reac-
ions we e incuba ed a 37 °C, o 2 h and he p oduc s
we e hen isola ed by hyd ophobic ch oma og aphy in
SepPak C18 Classic Ca idge (Wa e s). The columns
we e washed wi h wa e and elu ed wi h 1 mL ace o-
ni ile, which was coun ed in a liquid scin illa ion coun-
e . The inco po a ion on endogenous subs a es, in he
absence o he accep o subs a e, was sub ac ed.
BCG challenge o HT1376 cells
Comme cial Connaugh BCG (ImmuCys , Sano i Pas-
eu SA, F ance) was suspended in PBS con aining
0.05% Tween 80 and s o ed a −80 °C. Be o e each
assay, BCG agg ega es we e disca ded by cen i uga ion
(300 × g o 5 min). To assess BCG in e naliza ion, bac-
e ia we e s ained wi h 2 μg/mL o 5-(and-6-)(((4-chlo -
ome hyl)benzoyl)amino) e ame hyl hodamine (CMTR,
In i ogen) o 2 h in cul u e medium, incuba ed wi h
HT1376
T
o HT1376
sT
cells in a 1:10 cell/bac e ia a io
o 2 h a 37 °C and analyzed by low cy ome y. To as-
sess cy okine sec e ion, HT1376
T
o HT1376
sT
cells
we e challenged wi h uns ained BCG o 2 h a 37 °C,
he medium was emo ed and he cells we e washed
wice wi h PBS and incuba ed wi h esh medium o
16 h. Condi ioned media we e used o cy okine analysis
and o challenge mac ophages, while cell pelle s we e
used o RNA ex ac ion and ansc ip omic analysis.
De e mina ion o cy okine concen a ion
The concen a ion o cy okines IL-1β, IL-2, IL-4, IL-6,
IL-8, IL-10, IL-12, IL-17, IFN-γand TNF-αwas mea-
su ed in a 96-well s ip pla e om a comme cial MIBA
ki (Bio-Rad), as ecommended by manu ac u e ’s in-
s uc ions. Fluo escence was ead in a Luminex 100 Bio-
Plex Liquid A ay Mul iplexing Sys em eade (Bio-Rad)
and he da a analyzed wi h he Bio-Plex Manage 5
so wa e (Bio-Rad).
Mac ophage p epa a ion and s imula ion
Mononuclea cells we e isola ed by Ficoll-Hypaque
densi y g adien cen i uga ion (GE Heal hca e) om he
pe iphe al blood o heal hy blood-dono s. Fo his use,
no s udy app o al was necessa y. The only au ho iza ion
equi ed was ha ob ained om he Blood Collec ion
Se ice o he Pizza di Hospi al in Bologna, I aly, which
keeps he igh s on dono s’blood samples. Mac ophages
we e ob ained by di e en ia ion o monocy es by cul u e
in RPMI 1640 (Sigma) medium supplemen ed wi h 20%
FCS, 2 mM L-glu amine and 100 μg/mL penicillin/
s ep omycin. A e 7 days, monocy e-de i ed mac o-
phages we e de ached wi h a cell sc ape and dispensed
in 24 well pla es a a cell densi y co esponding
app oxima ely o 50% o con luence. One day la e , mac-
ophages we e incuba ed wi h s anda d uncondi ioned
cul u e medium o wi h he media condi ioned by
HT1376
T
o HT1376
sT
cells ei he BCG-challenged (as
desc ibed abo e) o mock challenged. A e 2 h, he con-
di ioned media we e eplaced by esh medium, which
was collec ed 24 h la e and s o ed a −80 °C o he de-
ec ion o cy okines sec e ed by mac ophages.
H
2
O
2
ea men
Cells in exponen ial g ow h phase we e incuba ed in
se um- ee medium con aining 5 mM H
2
O
2
o 1 h. The
medium was hen eplaced wi h esh comple e medium
and he cells we e ha es ed and analyzed 24 h la e .
Mock- ea ed cells we e incuba ed as abo e wi hou
H
2
O
2
. The cy o oxic e ec o H
2
O
2
was de e mined by
coun ing he numbe o cells in six eplicas seeded in 6-
wells pla es. Rep esen a i e ields we e pho og aphed
wi h an in e ed phase con as mic oscope.
Whole ansc ip ome analysis by exp ession mic oa ay
To al RNA was isola ed by he guanidinium hiocyana e-
me hod [29] and con e ed o labelled single s and
cDNA (ssDNA) by he comme cial Whole T ansc ip
Exp ession ki (Ambion), acco ding o he manu ac-
u e ’s ins uc ions. Labelled ssDNA agmen s we e hy-
b idized in a Human T ansc ip ome A ay 2.0 o e nigh .
A e s aining wi h phycoe y h in-s ep a idin, luo es-
cence was ead in a GeneChip Scanne 3000 7G (A y-
me ix). A e s a is ical analysis (see below), a ay da a
we e unc ionally analyzed by he A ayS a 2.0 so -
wa e (DNASTAR) and h ough a li e a u e sea ch o he
biological oles o modula ed genes. Gene nomencla u e
ollowed he HUGO Gene Nomencla u e Commi ee
ules (h ps://www.genenames.o g/) in i alic uppe case
le e s. Wi h excep ion o cy okines, p o eins had he
same name as he gene, ep esen ed in egula
uppe case.
S a is ical me hods
Mic oa ay aw da a we e backg ound-sub ac ed, no -
malized and summa ized wi h he obus mul i-a ay
a e age (RMA) algo i hm implemen ed in he A y pack-
age o Bioconduc o (www.bioconduc o .o g), which u i-
lizes R so wa e. Di e en ially exp essed genes be ween
que y and con ol assay we e selec ed by applica ion o
he wo ail ANOVA, ollowed by he Benjamini-
Hochbe g alse disco e y a e es wi h a po q≤0.05
cu -o and by he log
2
exp ession a io, conside ing only
a ia ions ≥0.5. MIBA da a we e analyzed by ANOVA,
ollowed by Tukey mul iple compa ison es . H
2
O
2
ox-
ici y da a we e analyzed by he S uden ’s es . The so -
wa e used was G aphpad P ism, e sion 7.0.
Se e ino e al. BMC Cance (2018) 18:198 Page 3 o 9
Resul s
ST3GAL1-exp ession leads o eplacemen o T wi h sT
an igen in HT1376 cells
The mock- ansduced cell line HT1376
T
, like he wild
ype HT1376, exp essed high and homogeneous T ex-
p ession, while he ST3GAL1- ansduced cell line
HT1376
sT
displayed an homogeneous low PNA eac i -
i y which could be e e ed o high eac i i y a e sialid-
ase ea men (Addi ional ile 1B). Bo h he ST3GAL1
ac i i y (Addi ional ile 1C) and he ST3GAL1 mRNA
measu ed by eal ime PCR (Addi ional ile 1D) we e
e y high in HT1376
sT
al hough almos unde ec able in
HT1376
T
. The le el o ST3GAL1 ac i i y and PNA e-
ac i i y eached by HT1376
sT
cells was simila o ha
displayed by he wild ype BC cell line 5637 s ongly ex-
p essing ST3GAL1 (da a no shown [24]). This is consis -
en wi h he no ion ha , in BC cells, ST3GAL1-media ed
α2,3 sialyla ion o galac ose (Addi ional ile 1A) masks he
Tan igen[24].
IL-6 and IL-8 sec e ion by HT1376
T
and HT1376
sT
cells
To assess he e ec o ST3GAL1 o e exp ession and he
consequen eplacemen o he T wi h he sT an igen on
cy okine p oduc ion ollowing BCG s imula ion, we
challenged he wo HT1376 cell lines wi h BCG. Among
he se e al cy okines es ed (IL-1β, IL-2, IL-4, IL-6, IL-8,
IL-10, IL-12, IL-17, IFN-γand TNF-α), only IL-8 was de-
ec able in media condi ioned by unchallenged HT1376
T
o
HT1376
sT
cells. A e BCG challenge, IL-6 became de ec -
able in bo h HT1376
sT
and HT1376
T
cells, while he IL-8
sec e ion showed a clea endency o up- egula ion a e
BCG challenge, especially in HT1376
T
cells (Fig. 1a). The p
alues a e epo ed in Addi ional ile 2.
To es ablish whe he he di e en IL-8 p oduc ion by
HT1376
sT
a e BCG challenge was due o a highe BCG
up ake by hese cells, we measu ed BCG in e naliza ion
by he wo HT1376 cell popula ions by low cy ome y.
As shown in Fig. 1b, he e we e no signi ican di e -
ences in he up ake o luo escen BCG by HT1376
T
and
HT1376
sT
cells, uling ou he possibili y ha di e ences
in cy okine p oduc ion by BCG-s imula ed HT1376
T
and
HT1376
sT
was dependen on di e en BCG in e naliza-
ion a e.
HT1376
sT
cells induce a s onge mac ophage sec e o y
esponse a e BCG s imuli
To in es iga e he ole played by ST3GAL1 exp ession
and he consequen T/sT eplacemen on he s imula-
ion o inna e immuni y by BCG-challenged BC cells,
he sec e ion o cy okines by human mac ophages s im-
ula ed wi h he sec e ome o BCG-challenged BC cells
was measu ed. Uns imula ed mac ophages sec e ed high
le els o IL-8 and e y low le els o TNF-α, IL-6, IL-1β
and IL-10. This pa e n o sec e ion was no signi ican ly
changed by s imula ion wi h he sec e ome o BCG-
unchallenged HT1376
T
o HT1376
sT
cells (Fig. 2). In
con as , he sec e ome om bo h BCG-challenged cell
lines, in pa icula ha om HT1376
sT
, signi ican ly in-
c eased he sec e ion o IL-6, IL-1β, TNF-αand IL-10
whe eas IL-8 sec e ion was ha dly a ec ed by he sec e-
ome o BCG-challenged cells, ega dless o hei
ST3GAL1 exp ession (Fig. 2). I should be no ed ha
he le el o sec e ion o IL-8 and IL-6 by mac ophages
was abou 10 and 3- old highe , espec i ely, han he
le el o ei he in HT1376 cells (Fig. 1), uling ou he
possibili y o a signi ican con amina ion o cy okines se-
c e ed by mac ophages wi h hose sec e ed by HT1376
cells. The cy okines IL-2, IL-4, IL-12 and IL-17 we e no
sec e ed by mac ophages in any o he es ed condi ions
(da a no shown). The pand q alues a e epo ed in
Addi ional ile 2.
HT1376
sT
cells display down- egula ion o genes
p ese ing genomic s abili y
The impac o ST3GAL1 o e exp ession and o T e-
placemen by sT on he global ansc ip ional ac i i y o
HT1376 cells was e alua ed by exp ession mic oa ay
echnology. O he 254 genes, which displayed
ST3GAL1-dependen modula ion by a leas a log
2
ex-
p ession a io ≥1.0 (meaning a old change o a leas 2)
wi h po q alues ≤0.05, 106 we e up- egula ed and 148
we e down- egula ed. The comple e lis o genes
BA
Fig. 1 BCG-s imula ion o HT1376 cells. a: Sec e ion o IL-6 and IL-8.
HT1376
T
, (whi e ci cles) and HT1376
sT
(black ci cles) cells we e challenged
wi h BCG o 2 h, hen hey we e washed and cul u ed in medium o
16 h. Cy okine concen a ion was de e mined as desc ibed in Ma e ials
and Me hods sec ion. Da a o h ee independen expe imen s a e
epo ed. S a is ical analysis is epo ed in Addi ional ile 2.b:Timecou se
o BCG up ake. HT1376T (whi e ci cles) and HT1376sT (black ci cles) cells
we e challenged wi h CMTMR-labelled BCG o 2 and 6 h, as desc ibed
in he Me hods sec ion. BCG in e naliza ion was es ima ed as mean
luo escence in ensi y o he cells, measu ed by low cy ome y Da a
o h ee independen expe imen s a e epo ed
Se e ino e al. BMC Cance (2018) 18:198 Page 4 o 9
showing signi ican modula ion (changes equal o highe
han 2- old) in HT1376
sT
s. HT1376
T
is epo ed in
Addi ional ile 3. A leas 29 genes pu a i ely egula ing
malignancy because o hei in ol emen in he con ol
o apop osis, cell g ow h, angiogenesis, in lamma ion o
p o eolysis, we e di e en ially exp essed in HT1376
sT
cells, compa ed wi h HT1376
T
cells (Addi ional ile 4). A
sea ch in he li e a u e e ealed ha 18 genes we e mod-
ula ed owa ds inc eased malignancy and 14 owa ds de-
c eased malignancy (Addi ional ile 4). Rema kably,
among he 18 genes modula ed owa ds inc eased malig-
nancy in HT1376
sT
cells, 11 down- egula ed genes can
be collec i ely e e ed o as “ca e ake genes”because
o hei in ol emen in DNA epai and mi o ic ideli y
(Addi ional ile 4). Mo eo e , i h eshold was lowe ed o
include genes down- egula ed abo e 1.3- old, he num-
be o down- egula ed ca e ake genes ose o 34
(Table 1). As a unc ional alida ion o gene exp ession
da a, cells we e exposed o he geno oxic e ec s o he
oxidizing agen H
2
O
2
. As expec ed o a cell line wi h
impai ed DNA epai mechanisms, H
2
O
2
- ea men
Fig. 2 Cy okine sec e ion by human mac ophages ea ed wi h
condi ioned media o BC cell lines. Cy okine sec e ion was measu ed
in cul u e media condi ioned by uns imula ed mac ophages (MΦ,
g ey ci cles) o s imula ed wi h condi ioned media om BCG-
challenged o unchallenged BC cell lines: HT1376
T
, (whi e ci cles);
HT1376
sT
(black ci cles). Cells we e challenged wi h BCG o 2 h, hen
hey we e washed and allowed o condi ion he cul u e medium o
16 h. The condi ioned cul u e media o BCG-challenged o unchallenged
cells was used o s imula e human monocy e-de i ed mac ophages o
24 h. MØ: cy okines eleased by uns imula ed mac ophages; MØ +
unchallenged: cy okines eleased by mac ophages s imula ed wi h
he condi ioned medium o unchallenged HT1376 cells; MØ + BCG-
challenged: cy okines eleased om mac ophages s imula ed wi h he
condi ioned medium o BCG-challenged cells. Da a o h ee independen
expe imen s a e epo ed. S a is ical analysis is epo ed in
Addi ional ile 2
Table 1 Genes in ol ed in main aining ch omosomal s abili y
and/o DNA epai showing down- egula ion in HT1376
sT
cells
Genes showing a old change ≥2 a e boxed in g ay
Se e ino e al. BMC Cance (2018) 18:198 Page 5 o 9

esul ed in a educed iabili y in HT1376
sT
han in
HT1376
T
cells (Fig. 3a and b).
HT1376
sT
cells display inc eased ansc ip omic changes
a e BCG challenging
To in es iga e he e ec o a di e en ial T/sT exp ession
on BCG challenging, he global ansc ip ional ac i i y
was also compa ed in BCG-challenged HT1376
T
and
HT1376
sT
cell lines. While in HT1376
sT
cells a g oup o
38 genes showed signi ican modula ion by BCG (pand
q alues ≤0.05) wi h a log
2
exp ession a io ≥0.5 ( old
change a leas 1.4) (Addi ional in o ma ion 5), he en-
dency o modula ion showed by a ew genes in HT1376
T
cells ne e eached s a is ical signi icance (da a no
shown). Modula ed genes in HT1376
sT
cells ell in o se -
e al b oad unc ional ca ego ies (Addi ional in o ma ion
5). In pa icula , a se o genes ela ed o “aminoacid and
p o ein biosyn hesis”we e p e alen ly down- egula ed in
BCG-challenged HT1376
sT
cells (Addi ional ile 5). This
unc ional ca ego y includes genes in ol ed in he biosyn-
hesis and anspo o aminoacids, a achmen o aminoa-
cids o RNAs and egula ion o ansla ion. O he
ca ego ies o modula ed genes include “cell g ow h”and
“in lamma o y and immune esponse”(Addi ional ile 5).
Discussion
The o e exp ession o ST3GAL1 has been widely s ud-
ied in cellula models o b eas cance and ound o be
esponsible o inc eased malignancy [30–32]. In con-
as , e y li le is known on he ole o his enzyme in
BC biology. In his wo k, we ha e pe o med an exhaus -
i e analysis o a BC expe imen al sys em in which
h ough he o e exp ession o ST3GAL1, he cons i u-
i ely exp essed T an igen was eplaced by i s sialyla ed
coun e pa , he sT ca bohyd a e s uc u e. Bo h s uc-
u es a e biologically ac i e, being ecep o s ei he o
galec ins o siglecs, as well as o suga binding ecep-
o s o mic oo ganisms [33]. Thus, he pheno ypic
changes we epo a e ST3GAL1 o e exp ession could
be a ibu able ei he o he lack o T an igen o exp es-
sion o sT o bo h. The modula ion o he immune e-
sponse, in pa icula , he sec e ion o cy okines, is a well
known unc ional consequence o BCG in e ac ion wi h
BC cells [1,34,35]. Ou s udy con i ms ha BCG in-
duces IL-6 and IL-8 sec e ion by BC cells and shows a
endency o highe IL-8 sec e ion by BCG-s imula ed
cells exp essing sT. In BC pa ien s, plasma [36] and is-
sue [37] IL-6 le els a e ele a ed and associa ed wi h
umo p og ession and poo p ognosis. P e ious s udies
ha e indica ed ha BCG s imula es IL-6 p oduc ion in
u ine o pa ien s and BC cell lines [38], inducing non
apop o ic cell dea h [39]. Inc eased u ina y IL-8 le el
also co ela es wi h p og ession in BC pa ien s [40], al-
hough a high u ina y IL-8 le el a e BCG ins illa ion is
a p ognos ic ac o o success ul ou come [41,42]. Ou
da a indica e ha a e BCG challenge, only he sec e-
ion o IL-6 and, o a lesse ex en , IL-8 we e s imula ed
while he o he cy okines emained unde ec able. This is
consis en wi h clinical obse a ions epo ing ha a ew
cy okines, including IL-6 and IL-8, we e de ec able in
u ine a e a i s in a esical BCG adminis a ion, while
o he cy okines equi ed mul iple BCG ins illa ions [43].
A e BCG challenge, he sec e ome o BC cells ex-
p essing he sT s uc u e showed highe capaci y o
s imula e cy okine sec e ion by mac ophages, u he
suppo ing he no ion ha he sT an igen po en ia es he
BC esponse o BCG.
An in lamma o y en i onmen can ei he p omo e o
inhibi umo p og ession, consis en wi h he no ion
ha in lamma ion is a double edge swo d which can
bo h e adica e he umo bu also uel i s g ow h. Mac-
ophages a e di e en ia ed ei he as p o-in lamma o y
M1 o an i-in lamma o y M2 pheno ypes, e en i in
many cases hey a e in an in e media e condi ion. While
a a iable capaci y o sec e e IL-6, IL-1βand TNF-αis
sha ed by di e en ially pola ized mac ophages, an IL-12
A
B
Fig. 3 Cy o oxic e ec o H
2
O
2
on HT1376
T
and HT1376
sT
cells. a: cells
we e ea ed wi h 5 mM H
2
O
2
o mock- ea ed as desc ibed in Me hods
and coun ed. Da a o six expe imen s a e epo ed. The HT1376
sT
cells
a e mo e suscep ible o he oxic e ec o H
2
O
2
(**p= 0.005 acco ding o
S uden ’s es ). b: phase con as mic og aph o ep esen a i e ields o
he wo cell popula ions ea ed as desc ibed abo e, showing he
s onge cy o oxic e ec s o H
2
O
2
on HT1376
sT
. Scale ba : 100 μm
Se e ino e al. BMC Cance (2018) 18:198 Page 6 o 9
low/IL-10 high pheno ype is he hallma k o M2 mac o-
phages [44], which a e known o p omo e umo g ow h
[45]. The mac ophages used in his s udy sec e ed IL-6,
IL-8, IL-1βand TNF-αbu no IL-12 and li le IL-10 and
a e p obably ep esen a i e o an in e media e condi ion
be ween he wo ex eme pheno ypes. The na u e o he
mac ophages associa ed wi h BC is indeed a iable as in-
dica ed by he di e en le el o exp ession o he M2-
speci ic ma ke CD163 among pa ien s [46]. In e es -
ingly, he p edominance o M2 mac ophages is associ-
a ed wi h highe s age and g ade [46] and wi h a wo se
esponse o BCG [47].
The mos p ominen ansc ip omic change we ob-
se ed because o ST3GAL1 exp ession and he conse-
quen eplacemen o he T wi h he sT an igen in
HT1376 cells was he dec eased exp ession o se e al
genes in ol ed in di e en mechanisms o DNA epai
and in he accu acy o ch omosomal seg ega ion. This
esul ed in inc eased sensi i i y o HT1376
sT
cells o he
cy o oxic e ec o H
2
O
2.
This is o in e es i one con-
side s ha he gene a ion o eac i e oxygen species by
BCG is a c ucial mechanism o BCG-induced damage o
BC cells [48]. In e es ingly, also in glioma cells,
ST3GAL1 exp ession esul ed in ansc ip omic changes
a ec ing malignancy and he cell cycle [49], suppo ing
he no ion ha a ca bohyd a e s uc u e on he cell su -
ace can gene a e an “ou side in” low o in o ma ion
modula ing gene exp ession [20].
BCG challenge esul ed in a deepe modula ion o he
ansc ip ome in HT1376
sT
han in HT1376T, sugges -
ing ha he eplacemen o he T wi h he sT an igen on
he cell su ace changes he de elopmen o he gene ic
p og am igge ed by BCG con ac .
E en hough genes in ol ed in in lamma o y and im-
mune esponse we e ound o be modula ed by BCG
challenge in HT1376sT cells, li le o no changes we e
obse ed in genes encoding cy okines, including hose
whose exp ession was s imula ed by BCG-challenge. Pos-
sible disc epancies be ween gene and p o ein exp ession
can be explained conside ing ha mul iple mechanisms op-
e a ing a pos ansc ip ional and pos ansla ional le els (in-
cluding non-coding RNAs, ansla ion egula o y
mechanisms, p o easomal ac i i y) egula e p o ein exp es-
sion. In addi ion, as shown o IL-1β, cy okine sec e ion
can be egula ed by he elease o p e o med molecules
om in acellula s o es, a he han a he le el o gene
ansc ip ion [50]. The soluble ac o s esponsible o he
s imula ion o cy okine sec e ion by mac ophages a e con-
cei ably a e y complex cock ail o bioac i e compounds o
p o ein and non-p o ein na u e, including biologically ac-
i e molecules (e.g. p os aglandins, glycosaminoglycans)
which a e no p ima y gene p oduc s bu p oduc s o mul-
iple enzyma ic eac ions. Fo hese easons, he na u e o
he molecules sec e ed by BC cells esponsible o he e ec
on mac ophages may no be di ec ly ela ed o he gene ex-
p ession p o ile.
Conclusions
In conclusion, ou da a show ha ST3GAL1 exp ession
and he consequen eplacemen o he T by he sT an i-
gen in BC cells induce ansc ip omic changes wi h a
pu a i e impac on mul iple cellula unc ions associa ed
wi h inc eased malignancy and suscep ibili y o oxida i e
damage and s eng hens he in lamma o y esponse o
mac ophages. This indica es ha ST3GAL1 and T/sT
ca bohyd a e s uc u es can be ac o s wi h mul iple
clinical implica ions in BC.
Addi ional iles
Addi ional ile 1: Figu e S1. ST3GAL1-o e exp ession in HT1376 cells
(PPTX 130 kb)
Addi ional ile 2: p alues calcula ed wi h ANOVA, ollowed by Tukey
mul iple compa ison es o da a epo ed in Fig. 1and Fig. 2(PPTX 84 kb)
Addi ional ile 3: GENES MODULATED IN HT1376sT CELLS AS
COMPARED TO HT1376T CELLS. (XLSX 47 kb)
Addi ional ile 4: CANCER-ASSOCIATED GENES MODULATED IN
HT1376sT CELLS AS COMPARED TO HT1376T CELLS. (XLS 43 kb)
Addi ional ile 5: GENES MODULATED BY BCG IN HT1376
sT
(XLS 49 kb)
Abb e ia ions
BC: bladde cance ; BCG: Bacillus Calme e-Gué in; CMTMR: 5-(and-6)-(((4-
chlo ome hyl)benzoyl)amino) e ame hyl hodamine; DMEM: Dulbecco’s
modi ied Eagle medium; FCS: Foe al cal se um; FITC: Fluo escein
iso hiocyana e; Gal: Galac ose; GalNAc: N-ace ylgalac osamine; GlcNAc:
N-ace ylglucosamine; MES: (N-mo pholino)e hanesulphonic acid;
MIBA: Mul iplex immune-beads assay; NMIBC: Non-muscle in asi e bladde
cance ; PBS: Phospha e-bu e ed saline; PNA: Peanu (A achis hypogea)
agglu inin; RPMI: Roswell Pa k Memo ial Ins i u e; Sia: sialic acid
Acknowledgemen s
We hank D . F ancesca Bo se i and D . Enzo Spisni, BIGEA Depa men o
he Uni e si y o Bologna o he help wi h he mul iplex immune-beads
assays, D . Ch is ine M. Be s o he c i ical eading o he manusc ip and D .
Ma ia Le izia Bacchi-Reggiani (DIMES) o ad ices in s a is ical analysis.
Funding
This wo k was suppo ed by: Po uguese Founda ion o Science and
Technology (FCT) PhD g an s SFRH/BD/45120/2008 (Paulo F. Se e ino), SFRH/
BD/81860/2011 (Ma iana Sil a) and SFRH/BD/100970/2014 (Mylène Ca ascal),
Liga Po uguesa Con a o Canc o 2011 (Mylène A. Ca ascal); P émio
Bluepha ma Ino ação Uni e sidade de Coimb a and he San ande To a/
Uni e sidade NOVA de Lisboa p izes (Paula Videi a). G an s om he
Uni e si y o Bologna (Fabio Dall’Olio). Ma iangela Ca e a and Giulia Ven u i
a e PhD s uden s suppo ed by g an s om he Uni e si y o Bologna.
A ailabili y o da a and ma e ials
The da ase s gene a ed du ing he cu en s udy a e a ailable om he
co esponding au ho on easonable eques .
Au ho s’con ibu ions
P.F.S and M.S. pe o med he bulk o he expe imen al wo k, analysed da a
and helped w i ing he manusc ip . M. Ca , G.V., M. Ca and R.B.F. pe o med
pa o he expe imen al wo k. N.M. and M. Chi . con ibu ed o he
es ablishmen o ansduced cell lines. A.A. pe o med mic oa ay
expe imen s and analysed mic oa ay da a. P.A.V. and F.D concei ed he
wo k, analysed he da a and w o e he manusc ip . All au ho s e ised
c i ically he manusc ip , ha e gi en inal app o al o he e sion o be
Se e ino e al. BMC Cance (2018) 18:198 Page 7 o 9
published and ag ee o be accoun able o all aspec s o he wo k in
ensu ing ha ques ions ela ed o he accu acy o in eg i y o any pa o
he wo k a e app op ia ely in es iga ed and esol ed.
E hics app o al and consen o pa icipa e
This s udy does no use samples om diseased pe sons. The blood used o
he p epa a ion o monocy e-de i ed mac ophages was om heal hy blood
dono olun a ies. Fo his, no s udy app o al was necessa y. The only
au ho iza ion equi ed was ha ob ained om he Blood Collec ion Se ice
o he Pizza di Hospi al in Bologna, I aly, which keeps he igh s on dono s’
blood samples. This au ho iza ion is a ailable o he Edi o .
Consen o publica ion
No applicable.
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
Publishe ’sNo e
Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in
published maps and ins i u ional a ilia ions.
Au ho de ails
1
Cen o de Es udos de Doenças C ónicas, CEDOC, NOVA Medical School/
Faculdade de Ciências Médicas, Uni e sidade NOVA de Lisboa, Campo dos
Má i es da Pá ia, 130, 1169-056 Lisbon, Po ugal.
2
UCIBIO, Depa amen o
Ciências da Vida, Faculdade de Ciências e Tecnologia, Uni e sidade NOVA de
Lisboa, 2829-516 Capa ica, Po ugal.
3
Dipa imen o di Medicina Specialis ica,
Diagnos ica e Spe imen ale, Sede di Pa ologia Gene ale, Uni e si à di
Bologna, Via S. Giacomo 14, 40126 Bologna, I aly.
4
Cen o In e dipa imen ale
Rice che sul Canc o “Gio gio P odi”, Uni e si à di Bologna, Bologna, I aly.
Recei ed: 3 Ap il 2017 Accep ed: 8 Feb ua y 2018
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